CAREER: Engaging high school students in the identification of genes that control skeletal growth and proportion

为什么人类有短趾骨和长腿骨，在挖掘、飞行、奔跑和游泳的物种中，骨骼比例是如何变化的？骨骼伸长所需的大多数基因突变都会产生比例矮小的骨骼。科学家们对骨骼的生长速度如何在局部上决定比骨骼中的其他骨骼更快或更慢知之甚少。通过比较骨骼非常不同的两种啮齿动物--老鼠和脚极长的两足跳鼠--的基因表达差异，库珀实验室确定了一些极好的候选基因，可以控制物种内和物种之间的生长速度差异。在这个NSF职业奖中详细介绍的项目将利用在发育中的鸡骨架中快速和轻松地过度表达基因的能力来测试这样的预测，即这些候选基因中的30个都足以增加或减少骨骼生长速度。这些候选基因尚不清楚控制骨骼发育的因素。可视化和分析这些被操纵的骨骼的方法非常适合让高中生参与实践研究体验。因此，该项目旨在为服务不足的社区中的数百名“高危”学生提供一个潜在的变革性学习机会，并扩大对骨骼生长和比例的遗传控制的知识。库珀实验室利用老鼠和两足跳鼠后肢比例的显著差异，它们前肢的相似性，以及相对较近的系统发育关系(最后一个共同祖先约在5500万年前)，通过RNA-Seq鉴定具有与相对生长速度密切相关的表达差异的基因。这些基因形成了一个预测的相互作用网络，高度丰富了已知的影响骨骼生长的途径(例如WNT、Notch和蛋白多糖信号)，尽管许多已鉴定的单个基因没有已知的生长板功能。他们将专注于一组30个基因，这些基因显示了肢体骨骼中模块化表达的最有力证据；基因表达在跳鼠脚加速伸长的进化过程中发生了变化，而在长得像老鼠的前肢中保持不变。为了确定调节骨骼生长速度的致病机制，实验室将在发育中的鸡翅骨骼中实施一个强大的逆转录病毒错误表达系统，以测试每个基因足以加速或抑制骨骼伸长的假设。这些目标将迅速扩展控制骨骼比例的分子工具包的已知内容，并将有助于绘制控制生长板特定基因表达的模块增强子，从而控制骨骼比例的发展和进化。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Why do humans have short toe bones and long leg bones, and how does skeletal proportion vary in species that dig, fly, run, and swim? Most mutations in genes that are required for bone elongation produce a proportionately dwarfed skeleton. Scientists know very little about how a bone's growth rate is locally determined to be faster or slower than other bones in the skeleton. By comparing gene expression differences in two rodents with very different skeletons, the mouse and bipedal jerboa with extremely long feet, the Cooper laboratory identified a number of genes that are excellent candidates to control growth rate differences within and between species. The project detailed in this NSF CAREER award will take advantage of the ability to quickly and easily over-express genes in the developing chicken skeleton to test the prediction that 30 of these candidate genes, which are not known to control skeletal development, are each sufficient to increase or decrease bone growth rate. The methods to visualize and analyze these manipulated skeletons are ideally suited to engage high school students in hands-on research experience. This project is therefore designed to provide a potentially transformative learning opportunity for hundreds of "at risk" students in an under-served community, in addition to expanding knowledge of the genetic control of skeletal growth and proportion. The Cooper laboratory has capitalized on the striking difference in hindlimb proportions of the mouse and bipedal jerboa, similarity of their forelimbs, and the relatively close phylogenetic relationship (last common ancestor ~55 million years ago) to identify genes by RNA-Seq with expression differences that robustly associate with relative growth rates. These genes form a predicted interaction network that is highly enriched for pathways that are known to influence skeletal growth (e.g. WNT, Notch, and proteoglycan signaling), though many of the individual genes that were identified have no known growth plate function. They will focus on a set of 30 genes that show the strongest evidence for modular expression in the limb skeleton; gene expression changed during the evolution of accelerated elongation of the jerboa foot and remained unchanged in the forelimb that grows similar to mouse. In order to identify causative mechanisms that modulate skeletal growth rate, the lab will implement a powerful retroviral mis-expression system in the developing chicken wing skeleton to test the hypothesis that each gene is sufficient to accelerate or inhibit skeletal elongation. The objectives will rapidly expand what is known of the molecular toolkit that controls skeletal proportion and will contribute to mapping modular enhancers that control growth plate-specific gene expression and thus the development and evolution of skeletal proportion.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.